development and validation of the uv- …

www.wjpps.com Vol 8, Issue 7, 2019.

675

Chaudhari et al. World Journal of Pharmacy and Pharmaceutical Sciences

DEVELOPMENT AND VALIDATION OF THE UV-

SPECTROPHOTOMETRIC AND RP-HPLC METHOD FOR

SIMULTENIOUS ESTIMATION OF ITRACONAZOLE AND

TERBINAFINE HYDROCHLORIDE IN BULK AND IN

FORMULATION

Prachi R. Chaudhari*, Dr. J. K. Patil, V. H. Jain and Dr. S. P. Pawar

Department of Quality Assurance, P.S.G.V.P.M’s College of Pharmacy, Shahada, Dist.

Nandurbar, 425409 Maharashtra.

Kavayitri Bahinabai Chaudhari Uttar Maharashtra Vidyapith, Jalgaon.

ABSTRACT

The main objective was develop and validate the UV-

Spectrophotometric and RP-HPLC method for the estimation

ofitraconazole and terbinafine hydrochloride in bulk and

pharmaceutical formulations as per ICH guidelines. An UV-

Spectrophotometric method for the quantitative determination of

itraconazole and terbinafine hydrochloride a highly potent antimycotic

in tablet was developed in present work. The parameters linearity,

precision, accuracy, limit of detection, limit of quantitationwere

studied according to ICH Guideline UV Spectroscopic determination

was carried out at an absorption maximum of 247 nm using methanol

as solvent. In the UV spectroscopic method linearity over the

concentration range of itraconazole was found to be 1-5µgm/ml with a correlation coefficient

0.9978. And for terbinafine hydrochloride was found to 2.5-10µgm/ml with a correlation

coefficient 0.9986. result of the analysis were validated statisticaly and by recoverybstudies.

The proposed method is simple, rapid, precise and accurate and can be used for the reliable

quantitation of itraconazole and terbinafine hydrochloride in pharmaceutical formulation. A

RP-HPLC method has been developed and validated to determine itraconazole and

terbinafine hydrochloride in tablet dosage form. The chromatography was performed on c18

column and a mobile phase consisting of methanol and 0.1% OPA in water(65:35) eluents

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.421

Volume 8, Issue 7, 675-702 Research Article ISSN 2278 – 4357

*Corresponding Author

Prachi R. Chaudhari

Department of Quality

Assurance, P.S.G.V.P.M's

College of Pharmacy,

Shahada, Dist. Nandurbar,

425409 Maharashtra.

Article Received on

25 April 2019,

Revised on 16 May 2019,

Accepted on 06 June 2019

DOI: 10.20959/wjpps20197-14076


676


where monitored by uv photo diode array detector at wawelength of 230 nm. The flow rate

was 1ml. The method was validated for linearity, precision, specificity, accuracy, Limit of

detection, limit of quantification and robustness.

KEYWORDS: UV-Spectrophotometric,RP-HPLC,Itraconazole,Terbinafine,Validation.

INTRODUCTION

Method validation is the process used to confirm that the analytical procedure employed for a

specific test is suitable for its intended use. Results from method validation can be used to

judge the quality, reliability and consistency of analytical results; it is an integral part of any

good analytical practice.

Analytic method development and validation are key elements of any pharmaceutical

development program. HPLC analysis method is developed to identify, quantity or purifying

compounds of interest. This technical brief will focus on development and validation

activities as applied to drug products. Drug analysis reveals the identification characterization

& determination of the drugs in mixtures like dosage forms & biological fluids. . HPLC is

one of the commonly used analytical techniques. HPLC can also be automated which

involve automated sampling, separation, detection, recording and calculation and printing of

results.

The basice of UV-visible spectrometry is the absorption of the UV- visible radiation, which

causes the electronic transition within the molecules by the radient energy of definite and

narrow wavelength of monochromatic radiations.

Itraconazole has a broader spectrum of activity than fluconazole (but not as broad as

horiconazole or posaconazole). In particular it is active against aspergillus which fluconazole

is not. it is also licenced for used in blastomycosis, sporotrichosis. Itraconazole is over 99%

protein bound and has virtually no penetration into cerebrospinal fluid.

Terbinafine, sold under the brand name Lamisil, among others is an antifungal medication

used to treat pityriasis versicolor, fungal nail infection, and ringworm including jock itch and

athlete’s foot. It is either taken by mouth or applied to the skin as a cream or ointment.

Tablets by mouth are often prescribed for treatment of onychomycosis. Terbinafine

hydrochloride may induce or exacervate subacute cutaneous lupus erythametosus.


677


Terbinafine is increasingly used in combination with itraconazole antifungal to treat resistant

or refractory mycoses due to synergistic in vitro antifungal activity. Due to its broad

antifungal spectrum, interest in terbinafine has expanded to include its use in range of

cutaneous and subcutaneous mycosis, such as sporotrycosis. As well as in combination with

itraconazole to treat resistant or refreactory invasive fungal infection.

EXPERIMENTATION

UV-specrophotometric evaluation

Chemicals and reagent: Methanol was used throughout UVspecrophotometric method

development and validation.

INSTRUMENTATION

UV-spectrophotometric method was performed on doble beam spectrometer having two

matched quartz cells within 1 cm light path.

Selection of solvent:- Methanol was selected as a suitable solvent for spectrophotometric

analysis of itraconazole and terbinafine.

Detection of wawelength

For itraconazole

Sample-1

Abs

orba

nce(

Abs

)

Wavelength(nm)

-1

0

1

2

3

4

5

200 250 300 350 400

(201

.0, 1

.354

2) (2

03.0

, 2.9

687)

(205

.0, 2

.185

5) (2

06.0

, 2.5

172)

(241

.0, 0

.658

0)


678


For itraconazole

Sample-1

Abs

orba

nce(

Abs

)

Wavelength(nm)

-1

0

1

2

3

4

5

200 250 300 350 400

(201

.0, 0

.438

1)

(222

.0, 2

.332

5)

(281

.0, 0

.198

2)

Isobastic point-

Sample-2

Abs

orba

nce(

Abs

)

Wavelength(nm)

-1

0

1

2

3

4

5

200 250 300 350 400

(201

.0, 2

.496

4)(2

02.0

, 1.9

433)

(203

.0, 2

.346

4)

(241

.0, 0

.659

9)


679


Preparation of standard stock solution:- accurately weighed quantity of 10mg of

itraconazole and 25mg of terbinafine standard was transferred into 100ml volumetric flask

and dissolved and diluted upto mark using 100ml methanol of 100µg/ml.

Preparation of sample stock solution:- Preparation of sample stock solution:- to measure

the content of tablet containing itraconzole and terbinfine, 14.45 gm powder was taken in 100

ml volumetric flask and add 100 ml of methanol then well mixed and filtered to produced

strength of 100µ gm/ml and 250 µgm/ml respectively .

VALIDATION OF UV-SPECTROPHOTOMETRIC METHOD

Linearity and range:- The linearity was determined by analyzing 5 independent levels of

calibration curve in the range of 1-5µgm/ml and 2.5-12.5µgm/ml of itraconazole and

terbinafiner respectively. absorbance of each solution against methanol was recorded at 247

nm 222nm respectively. The calibration curve of absorbance ver conc. Was plotted and

correlation co-efficient and regression line equation for itraconazole and terbinafine were

determined.

Precision:-intra-day precision was determined by analyzing itraconazole and terbinafine (2-4

µg/ml and 5-10 µg/ml respectively.) at 3 different points of the same day and interday

precision was determined by analyzing itraconazole and terbinafine (2-4 µg/ml and 5-10

µg/ml respectively) at 3 different time points on different days and %RSD was calculated.

Accuracy:- accuracy was detrmined by performing recovery studies by speaking different

conc. of pure drug in the preanalyse powder for infusion samples within the analytical conc.

range of the proposed method at 3 different set at level of 80%, 100%, and 120%. The

amount of itraconazole and terbinafine was calculated at each level and % recovery were

computed.

Repeatability:- it was determined by analyzing 4µgm/ml and 10 µg/ml conc of itraconazole

and terbinafine solution for 5 times respectively.

LOD and LOQ:- the LOD and LOQ were estimated from the set of 5 calibration curves used

to determine method of linearity.

LOD= 3.3×avg SD/ slope

LOQ=10×avg SD/ slope

Assay:- to measure the content of tablet containing itraconzole and terbinfine,the 20 tablets

give weight of 14.45 gm powder then calculate the average weight of tablet. Then 72.7 mg


680


weight of poder was taken in 100 ml volumetric flask and add 100 ml of methanol then

sonicate for 15 minute and filtered to produced strength of 100µgm/ml and 250 µgm/ml .

HPLC evaluation

The various chromatographic conditions were established by trial and error and were kept

constant throughout the experimentation. The HEWLETT PACKARD series 1100 HPLC

instrument was used.

CHROMATOGRAPHIC CONDITIONS

The following chromatographic conditions were established by trial and error and were

kept constant throughout the experimentation

HPLC : Younglin ( S.K) Gradient System UV Detector

Detecter & pump No. : UV 730 D & SP930 D

Software : Autochro -3000

Column : 4.6 x 250 mm

Particle size packing : 5 m

Stationary phase : C18 (AGILENT)

Mobile Phase :- Methanol : (0.1 % OPA IN WATER)

65 : 35 (mix pH-3.1)

Detection Wavelength : 230 nm.

Flow rate : 1.0 ml/min

Temperature : Ambient

Sample size : 20 l

MATERIALS AND METHODS

Materials:-

Ingredients Grade Suppliers

Itrconazole API R.S.I.T.C Jalgaon.

Terbinfine API R.S.I.T.C Jalgaon.

Orthophopsphoric

acid(OPA) HPLC

Avantor Performance material India Ltd.

Thane, Maharashtra

Methanol HPLC Merck Specialities Pvt. Ltd. Shiv Sager Estate

‘A’ Worli, Mumbai

Water HPLC Merck Specialities Pvt. Ltd.Shiv Sager Estate

‘A’ Worli, Mumbai


681


Methods

Preparation of standard stock solution:- accurately weighed quantity of 10mg of

itraconazole and 25 mg of terbinafine reference standard was transferred into 10ml

volumetric flask and dissolved and diluted upto mark using 10 ml methanol having strength

of 1000µg/ml and 2500 µg/ml respectively.

Preparation of sample stock solution:- to measure the content of tablet containing

itraconzole and terbinfine, the 20 tablets give weight of 14.45 gm powder then calculate the

average weight of tablet. Then 72.7 mg weight of poder was taken in 10 ml volumetric flask

and add 10 ml of methanol then well mixed and filtered to produced strength of 1000µgm/ml

and 2500 µgm/ml.

VALIDATION OF UV-SPECTROPHOTOMETRIC METHOD

Linearity and range:- the linearity was determined by analyzing 5 independent levels of

calibration curve in the range of 5-25µgm/ml and 12.5-62.5µgm/ml itraconazole and

terbinafine respectively. absorbance of each solution against methanol was recorded at

230nm. The calibration curve of absorbance ver conc. Was plotted and correlation co-

efficient and regression line equation for itraconazole and terbinafine were determined.

Precision:-intra-day precision was determined by analyzing itraconazole and terbinafine (10-

20µg/ml and 25-50µg/ml respectively.) at 3 different points of the same day and interday

precision was determined by analyzing itraconazole and terbinafine (10-20 µg/ml and 25-50

µg/ml respectively) at 3 different time points on different days and %RSD was calculated.

Accuracy:- accuracy was detrmined by performing recovery studies by speaking different

conc. of pure drug in the preanalyse powder for infusion samples within the analytical conc.

range of the proposed method at 3 different set at level of 80%, 100%, and 120%. The

amount of itraconazole and terbinafine was calculated at each level and % recovery were

computed.

Repeatability:- it was determined by analyzing 4µgm/ml and 10 µg/ml conc of itraconazole

and terbinafine solution for 5 times respectively.

LOD and LOQ:- the LOD and LOQ were estimated from the set of 5 calibration curves used

to determine method of linearity.

LOD= 3.3×avg SD/ slope

LOQ=10×avg SD/ slope


682


Robustness-change following parameters one by one and observe their effects on system

sutability test and assay

Change flow rate by ± 0.1

Change ratio of mobile phase by ± 1

Change wawelength by ±1

Assay:- to measure the content of tablet containing itraconzole and terbinfine,the 20 tablets

give weight of 14.45 gm powder then calculate the average weight of tablet. Then 72.7 mg

weight of poder was taken in 10 ml volumetric flask and add 10 ml of methanol then sonicate

for 15 minute and filtered to produced strength of 1000µgm/ml and 2500 µgm/ml.

RESULT AND DISCUSSION

Uv Spectrophotometer

Itraconazole

CONC. avg.area

1 0.12

2 0.22

3 0.29

4 0.38

5 0.47

Linearity :-

Y=0.086X+0.038

Sr No. Conc ABS-I ABS-II Mean SD % RSD

1 1 0.1253 0.1239 0.12 0.001 0.79

2 2 0.2167 0.2146 0.22 0.001 0.69

3 3 0.2917 0.2914 0.29 0.000 0.07

4 4 0.3811 0.3912 0.39 0.007 1.85

5 5 0.47 0.471 0.47 0.001 0.15


683


PRECESION

Intraday

Sr No. Conc ABS-I ABS-II Mean Amt Found % Amt Fnd SD RSD

1 2 0.2123 0.2124 0.21 2.02 101.00 0.00 0.03

2 3 0.2899 0.2911 0.29 2.93 97.86 0.00 0.29

3 4 0.392 0.3884 0.39 4.09 102.23 0.00 0.65

Interday

Sr No. Conc ABS-I ABS-II Mean Amt Found % Amt Fnd SD RSD

1 2 0.2123 0.2124 0.21 2.02 101.00 0.00 0.03

2 3 0.2899 0.2911 0.29 2.93 97.86 0.00 0.29

3 4 0.392 0.3884 0.39 4.09 102.23 0.00 0.65

SST 0R REPEATIBILITY

Repitability

Sr No. Conc ABS-I Amt Found %Amt Found

1 4 0.3917 4.11 102.81

2 4 0.3911 4.10 102.64

3 4 0.3901 4.09 102.35

4 4 0.3905 4.10 102.46

5 4 0.3913 4.10 102.70

Mean 4.10 102.59

SD 0.01 0.19

%rsd 0.17 0.18

%Lable Claim

Y=0.086X+0.038

SR NO. Conc ABS-I

I Amt Found % Label Claim

1 2.00 0.2098 1.99 99.88

2 2.00 0.211 2.01 100.58

Mean 0.21 24.85 100.23

SD 0.00 0.01 0.35

%RSD 0.40 0.06 0.37

Accuracy

80%

Sr no. conc Amt

added ABS-I Amt found

Amt

rcvd % rcvd

1 2 1.6 0.3463 3.58 1.58 98.75

2 2 1.6 0.3492 3.61 1.61 101.16

Mean 3.60 1.60 99.96

SD 0.02 0.02 1.70

%RSD 0.59 1.33 1.70


684


100%

Sr no. conc Amt

added ABS-I

Amt

found

Amt

recvd

%

Recv

1 2 2 0.3871 4.05 2.05 102.96

2 2 2 0.3861 4.04 2.04 102.38

Mean 4.05 2.05 102.67

SD 0.01 0.01 0.41

%RSD 0.17 0.35 0.40

120%

Sr no. conc Amt added ABS-I Amt found Amt rcvd % Rcvd

1 2 2.4 0.422 4.46 2.46 102.81

2 2 2.4 0.4226 4.472 2.47 103.00

Mean 4.47 2.47 102.91

SD 0.01 0.01 0.13

%RSD 0.19 0.29 0.13

TERBINAFINE

Linearity

CONC. avg.area

2.5 0.2

5 0.32

7.5 0.47

10 0.61

12.5 0.76

Linearity :-

Sr No. Conc ABS-I ABS-II Mean SD RSD

1 2.5 0.2011 0.2013 0.20 0.00 0.07

2 5 0.3211 0.322 0.32 0.00 0.20

3 7.5 0.4781 0.4736 0.48 0.00 0.67

4 10 0.619 0.621 0.99 0.00 0.14

5 12.5 0.76 0.7511 0.76 0.01 0.83


685


Precision

Intraday

Sr No. Conc ABS-I ABS-II III Mean Amt Found % Amt Fnd SD RSD

1 5 0.3321 0.3353 0.33 5.01 100.20 0.002 0.68

2 7.5 0.4715 0.4727 0.47 7.51 100.13 0.001 0.18

3 10 0.6158 0.6207 0.62 10.19 101.90 0.003 0.56

Interday

Sr No. Conc ABS-I ABS-II III Mean Amt Found % Amt Fnd SD RSD

1 5 0.3321 0.3353

0.33 5.01 100.20 0.002 0.68

2 7.5 0.4715 0.4727

0.47 7.51 100.13 0.001 0.18

3 10 0.6158 0.6207

0.62 10.19 101.90 0.003 0.56

Repitability

Sr No. Conc ABS-I Amt Found %Amt Found

1 10 0.620 10.19 101.96

2 10 0.613 10.07 100.78

3 10 0.615 10.10 101.07

4 10 0.616 10.12 101.25

5 10 0.62 10.19 101.96

Mean 10.13 101.40

SD 0.05 0.53

%rsd 0.53 0.53

% Lable Claim

SR NO. Conc ABS-I


1 5.00 0.3283 4.98 99.60

2 5.00 0.3298 5.01 100.20

Mean 0.33 4.99 99.90

SD 0.00 0.02 0.64

%RSD 0.32 0.43 0.61

Accuracy

80%

Sr no. conc Amt added ABS-I Amt found Amt rcvd % rcvd

1 5 4 0.5512 8.96 3.96 99.00

2 5 4 0.5515 8.97 3.97 99.33

8.97 3.97 99.17

SD 0.01 0.01 0.23 %RSD 0.08 0.18 0.24

100%

Sr no. Conc Amt added ABS-I Amt found Amt recvd % Recv

1 5 5 0.6124 10.06 5.06 101.21


686


2 5 5 0.6126 10.05 5.05 101.05

Mean 10.06 5.06 101.13

SD 0.01 0.01 0.11

%RSD 0.07 0.14 0.11

120%

Sr no. Conc Amt added ABS-I Amt found Amt rcvd % Rcvd

1 5 6 0.6656 11.01 6.01 100.17

2 5 6 0.665 11.00 6.00 100.00

Mean 11.01 6.01 100.09

SD 0.01 0.01 0.12

%RSD 0.06 0.12 0.12

HPLC CALCULATION

ITRACONAZOLE

CONC. avg.area

5 105.20

10 184.35

15 255.55

20 356.63

25 423.19

Linearity

Sr No. Conc AREA-I AREA-II Mean SD RSD

1 5 101.52 103.48 102.50 1.39 1.35

2 10 182.91 185.79 184.35 2.04 1.10

3 15 254.23 256.87 255.55 1.87 0.73

2 20 359.96 353.29 356.63 4.72 1.32

3 25 418.31 428.06 423.19 6.89 1.63

AVG 6.17


687


Precision

Interday

Sr No. Conc Area II Mean Amt

Found

% Amt

Fnd SD RSD

1 10 185.231 188.5 186.87 10.17 101.70 2.31 1.24

2 15 265.74 260.83 263.29 14.90 99.33 3.47 1.32

3 20 352.97 350.63 351.80 20.37 101.85 1.65 0.47

Repitability

Sr No. Conc Peak Area Amt Found %Amt Found

1 20 349.29 20.22 99.76

2 20 352.97 20.44 102.20

3 20 345.63 19.99 99.95

4 20 350.17 20.27 101.38

5 20 349.02 20.20 101.02

Mean 20.22 100.86

SD 0.16 1.02

%rsd 0.80 1.01

% Lable Claim

SR NO. Conc Area


1 250.00 425.08 24.91 99.64

2 25.00 427.04 25.03 100.12

Mean 426.06 24.85 99.88

SD 1.39 0.08 0.34

%RSD 0.33 0.34 0.34

Accuracy

80%

Ng/Band Amt

added Area

Amt

found

Amt

recvd

%

Recv

10 8 314.06 18.04 8.04 100.52

10 8 312.14 17.92 7.92 99.00

Mean 17.98 7.98 99.76

SD 0.08 0.08 1.07

%RSD 0.47 1.06 1.08

100%

Ng/Band Amt added Area Amt found Amt recvd % Recv

10 10 348.39 20.18 10.18 101.80

10 10 345.27 19.97 9.97 99.73

Mean 20.08 10.08 100.77

SD 0.15 0.15 1.46

%RSD 0.74 1.47 1.45


688


120%


10 12 380.72 22.16 12.16 101.39

10 12 377.16 21.94 11.94 99.55

Mean 22.05 12.05 100.47

SD 0.16 0.16 1.30

%RSD 0.71 1.29 1.29

ROUBUSTNESS

FLOW-

0.9 ML

FLOW CHANGE

0.8 ML

1.1

ML

Sr No. Conc

Area Sr

No. Conc

Area

1 15

291.98 1 15

245.53

2 15

295.43 2 125

242.34

Mean 293.71

Mean 243.94

SD 2.44

SD 2.26

%RSD 0.83

%RSD 0.92

Mobil phae

Volume : 66+34

64+36

Sr No. Conc

Area

Sr

No. Conc

Area

1 15

256.73 1 15

242.5

2 15

258.66 2 15

244.26

Mean 257.70

Mean 243.38

SD 1.36

SD 1.24

%RSD 0.53

%RSD 0.51

WAVELENGTH

CHANGE = 223 229

WAVE LENGTH

CHANGE =225 231

Sr No. Conc

Area

Sr

No. Conc

Area

1 15

249.56 1 15

272.68

2 15

246.7 2 15

274.56

Mean 248.13

Mean 273.62

SD 2.02

SD 1.33

%RSD 0.82

%RSD 0.49

LOD=

3.3 X Avd.SD/

Slope

3.3x6.17/16.16 = 1.2599

LOQ=

10X Avd

SD/Slope

10 x6.17/16.16= 3.8180


689


TERBINAFINE

CONC. avg.area

12.5 606.17

25 1036.6

37.5 1549.22

50 2013.15

62.5 2522.71

Linearity

Sr No. Conc AREA-I AREA-II

Mean SD RSD

1 12.5 611.4 600.93

606.17 7.40 1.22

2 25 1032.69 1040.51

1036.60 5.53 0.53

3 37.5 1540.91 1557.53

1549.22 11.75 0.76

2 50 2013.78 2012.51

2013.15 0.90 0.04

3 62.5 2521.21 2524.2

2522.71 2.11 0.08

AVG 5.54

Precision

Interday

Sr No. Conc Area II III Mean Amt Found % Amt Fnd SD RSD

1 25 1091.61 1094.2

1092.91 25.74 102.96 1.83 0.17

2 37.5 1527.43 1535.28

1531.36 37.13 99.01 5.55 0.36

3 50 2002.43 2027.83

2015.13 49.71 99.42 17.96 0.89

SST 0R REPEATIBILITY

Repitability

Sr No. Conc Peak Area Amt Found %Amt Found

1 50 2012.43 49.64 99.28

2 50 2002.43 49.38 98.76

3 50 2003.15 49.40 98.80

4 50 2038.54 50.32 100.64

5 50 2011.39 49.61 99.22

Mean 49.67 99.34

SD 0.38 0.76

%rsd 0.77 0.77


690


%Lable Claim

SR NO. Conc Area


1 62.50 2506.48 62.48 99.97

2 62.50 2509.49 62.56 100.10

Mean 2507.99 24.85 100.03

SD 2.13 0.06 0.09

%RSD 0.08 0.23 0.09

Accuracy

80%


25 20 1850.19 45.42 20.42 102.10

25 20 1841.02 45.18 20.18 100.94

Mean 45.30 20.30 101.52

SD 0.17 0.17 0.82

%RSD 0.37 0.84 0.81

100%


25 25 2040.41 50.37 25.37 101.48

25 25 2035.2 50.23 25.23 100.94

Mean 50.30 25.30 101.21

SD 0.10 0.10 0.38

%RSD 0.20 0.39 0.38

120%


25 30 2230.21 55.30 30.30 101.01

25 30 2216.09 54.93 29.93 99.79

Mean 55.12 30.12 100.40

SD 0.26 0.26 0.86

%RSD 0.47 0.87 0.86

ROUBUSTNESS

FLOW-0.9 ML

FLOW CHANGE

0.8 ML 1.1 ML

Sr No. Conc

Area Sr No. Conc

Area

1 37.5

1685.05 1 37.5

1302.67

2 37.5

1694.55 2 37.5

1292.41

Mean 1689.80

Mean 1297.54

SD 6.72

SD 7.25

%RSD 0.40

%RSD 0.56


691


Mobil phae Volume :

66+34

64+36

Sr No. Conc

Area Sr No. Conc

Area

1 37.5

1535.87 1 37.5

1476.9

2 37.5

1541.86 2 37.5

1459.65

Mean 1538.87

Mean 1468.28

SD 4.24

SD 12.20

%RSD 0.28

%RSD 0.83

WAVELENGTH

CHANGE = 223 229

WAVE LENGTH

CHANGE =225 231

Sr No. Conc

Area Sr No. Conc

Area

1 37.5

1558.28 1 37.5

1559.49

2 37.5

1557.67 2 37.5

1571.88

Mean 1557.98

Mean 1565.69

SD 0.43

SD 8.76

%RSD 0.03

%RSD 0.56

LOD=

3.3 X Avd.SD/

Slope

3.3x5.54 /38.47 =0.4752

LOQ=

10X Avd

SD/Slope

10 x5.564/38.47= 1.4463

HPLC ANALYSIS RESULTS

Sample: LIN 15+ 37.5 -01

Date: 2019-05-17

Chromatogram


692


Result

No. Name RT[min] Area[mV

*s]

Area% TP TF Resolutio

n

1 3.5167 256.8219 14.63 3047.9 1.2500 0.0000

2 5.4000 1557.537

7

85.37 3444.5 1.1667 5.1364

Sample: LIN 25+ 62.5 -02

Date: 2019-05-17

Chromatogram

Result

No. Name RT[min] Area[mV

*s]

Area% TP TF Resolutio

n

1 3.4833 428.0670 14.27 2990.4 1.2500 0.0000

2 5.3833 2524.202

1

85.73 3423.2 1.2273 5.1818

Sum

Sample: PRECESION 10 + 25-01

Date: 2019-05-17


693


Chromatogram

Result

No. Name RT[min] Area[mV*s] Area% TP TF Resolution

1 3.5000 185.2185 14.51 2445.4 1.2500 0.0000

2 5.3667 1091.6145 85.49 3402.1 1.2083 4.8696

Sum 1276.8330

Sample: SST 20+ 50 -01

Date: 2019-05-17

Chromatogram


694


Result


1 3.5000 349.2918 14.19 3019.1 1.1875 0.0000

2 5.3833 2012.5149 85.81 3423.2 1.1667 5.1364

Sum

Sample: TAB ASSAY 25+ 62.5 -01

Date: 2019-05-17

Chromatogram

Result


1 3.4833 439.2149 14.10 2990.4 1.1875 0.0000

2 5.3667 2506.4814 85.90 3402.1 1.1250 5.1364

Sum

Sample: TAB ASSAY 25+ 62..5 -02

Date: 2019-05-17


695


Chromatogram

Result


1 3.3500 441.0501 14.10 2765.8 1.1875 0.0000

2 5.2167 2509.4983 85.90 3214.5 1.1250 5.0909

Sum 3270.5483

Sample: Accuracy 80% -01

Date: 2019-05-17

Chromatogram


696


RESULT


1 3.4667 319.0600 14.14 2961.8 1.1875 0.0000

2 5.3000 1850.1943 85.86 3318.1 1.2273 5.0000

Sum


Date: 2019-05-17

Chromatogram

Result


1 3.2500 322.1452 14.15 2108.6 1.1111 0.0000

2 5.0500 1841.0281 85.85 3012.4 1.2273 4.6957

Sum


Date: 2019-05-17


697


Chromatogram

Result


1 3.3167 348.7294 14.10 2711.1 1.1875 0.0000

2 5.1167 2040.2424 85.90 3092.5 1.2273 4.9091

Sum


Date: 2019-05-17

Chromatogram


698


Result


1 3.3667 345.273 14.11 2793.4 1.1875 0.0000

2 5.1500 2035.2080 85.89 3132.9 1.2273 4.8636

Sum


Date: 2019-05-17

Chromatogram

Result


1 3.4167 380.7294 14.28 2877.0 1.1875 0.0000

2 5.1833 2230.2100 85.72 3173.6 1.1818 4.8182

Sum


Date: 2019-05-17


699


Chromatogram

Result


1 3.4333 377.1693 14.02 2905.1 1.1875 0.0000

2 5.2000 2216.0962 85.98 3748.6 1.1818 5.0476

Sum

Sample: ROBUSTNESS COMP CHANGE 66+34-002

Date: 2019-05-17

Chromatogram

Result


1 3.5833 258.6636 14.37 2563.3 1.1667 0.0000


700


2 5.4500 1541.8698 85.63 3025.2 1.1667 4.6667

Sum 1800.5334

Sample: Roubustness Wave Length Change 231 mcg-01

Date: 2019-05-17

Chromatogram

Result


1 3.4167 272.6828 14.20 2330.4 1.1875 0.0000

2 5.2333 1559.4968 85.80 3235.1 1.1250 4.7391

Sample: 25mcg ITRACONAZOLE

Chromatogram


701


Result


1 3.4833 428.0670 100.00 2990.4 1.2500 0.0000

Sum

Sample: 62.5 MCG TERBINAFINE

Chromatogram

Result


1 5.3833 2524.2021 100.00 3423.2 1.2273 0.0000

CONCLUSION

This UV- spectrophotometric technique is quite simple, accurate, precise, reproducible and

sensitive. The UV method has been developed for quantification of simultaneous estimation

of itraconazole and terbinafine in powder formulation. The validation procedure confirms that

this is an appropriate method for their quantification in the formulation. It is also used in

routine control of the formulations containing this entire compound. RP-HPLC are found to

be more precise, accurate, robust. All this developed method can be use for routine analysis

of itraconazole and terbinafine in pharmaceutical formulation.

REFERENCES

1. https”//www.medicinenet.com.

2. https://en.m.wikipedia.org>wiki>itraconazole.


702


3. Kaelgren M. vildhede A, norinder U, :classification of inhibitors of hepatic organic anion

transporting polypeptides : influence of protein expression on drug –drug interaction.

4. www.life-worldwidw.org.

5. Preissner S, a comprehensive database on cytochrome P450 enzymes includind a tool for

analysis of CYP- drug interaction nucleic acids.

6. https://pubchem.ncbi.n/m.nih.gov terbinafine.

7. https://en.m.wikipedia.org Wikipedia. Org.

8. Roberts so. Pityriasis versicolor: a clinical and mycological investigation.

9. https;//www.acessdata.fda.gov>label SPORANOX (itraconazole) FDA.

10. https;//www.acessdata.fda.gov>label Lamisil (terbinafine hydrochloride tablet) ICH-

guidwlines Q2 B validation of analytical procedures methodology. Geneva, November,

1996; (CPMP/ICH/281/95), 1-10.

11. ICH topic Q1 A (R2) stability testing of new drug substances and product. GENEVA

august, 2003. (CPMP/ICH/2736/99)

12. AL-ravithi H, Sameer M, husain A, raja almoshen, Ibrahim, raines, dale, etal.

deatermination of itraconazole and hydroxyl itraconazole in plasma by HPLC with

fluorescence detection, therapeutic drug monitoring, 2001: 445-448.

13. Cardos, elefrides E.S. schepovul, HPLC assay of terbinafine hydrochloride in tablets and

creams, Jpharm biomade analysis, 1999; 809-812.

http://www.life-worldwidw.org/

https://pubchem.ncbi.n/m.nih.gov

https://en.m.wikipedia.org/

development and validation of the uv- …

Documents